Casein phosphopeptide salts

ABSTRACT

Casein phosphopeptide salts of calcium, magnesium or both are produced having less than 4% by weight of aromatic amino acids, greater than 8% and less than 20% by weight serine, less than 3% free amino acid content and a ratio of total calcium, magnesium and phosphorus to total nitrogen greater than 0.2. The phosphopeptides are produced by subjecting a casein material to proteolytic enzyme hydrolysis ultrafiltering the resulting hydrolyzate to produce a permeate containing phosphopeptides, adding a bivalent cation salt to the peptides to form phosphopeptide aggregates and separating by ultrafiltration the phosphopeptide aggregates from non phosphorylated peptides. The phosphopeptides obtained are useful as aliments for dietetic or therapeutic nutrition and as medicines.

The present application is a continuation of Ser. No. 07/227,515, filedon Aug. 2, 1988, now abandoned; which is a continuation of Ser. No.096/820,840, filed on Jan. 22, 1986, now U.S. Pat. No. 4,816,398; whichis a continuation of Ser. No. 388,931, filed on June 16, 1982, now U.S.Pat. No. 4,495,176; which is a continuation of Ser. No. 229,062, filedJan. 28, 1981, now U.S. Pat. No. 4,358,465.

The present invention relates to the field of processing casein-basedmaterials. More particularly, it has for its object a method involvingtreatment of phosphocaseinates of monovalent cations or derivativesthereof. The invention also relates to the products obtained by such amethod, especially fractions-enriched in phosphopeptides and in nonphosphorylated peptides, respectively. The invention further relates tothe applications of the thus obtained products, particularly asalimentary products adapted to meet specific nutrition requirements aswell as medicaments.

It is known that the caseins of dairy raw materials, and mostparticularly of milk, contain phosphoserines which impart to thepeptides wherein they occur valuable physico-chemical, technological andphysiological properties. Among others, information on milk proteinswill be found in the book by Mc KENZIE H. A. (1971) entitled "Milkproteins", Vol. 1 and 2, Academic Press, New York.

Due to the recent advancement as regards both the apparatuses and theunderstanding of the observed phenomena, membrane ultrafiltration hasfound broad acceptance in the milk industry, for milk treatment [see,for example, Maubois J. L. Mocquot G. (1971-Preparation de fromages apartir de pre-fromages liquides obtenus par ultrafiltration du lait "LELAIT", fascicule 51 508, 495-533)]. As the milk flows across theultrafiltration membrane, the water, soluble mineral salts, lactose, lowmolecular weight nitrogenous compounds (peptides, free amino-acids) andwater-soluble vitamins will pass through the membrane as anultrafiltrate or permeate, while the proteins and associated components(calcium, phosphorus), fat globules and lipophilous elements will beretained, their concentration increasing as the aqueous phase removalproceeds; these form the retentate or proteic concentrate. Obtention ofhigh purity proteic concentrates requires both an ultrafiltration stepand a diafiltration step. In the diafiltration step, addition of wateror aqueous solution containing salts is effected, in continuous ordiscontinuous manner, into the ultrafiltration retentate. Simultaneouslyor subsequently, an equivalent amount of permeate is removed. The resultof such operation is to deplete the amount of filterable elements in theretentate. The advantage of the membrane ultrafiltration technique is tokeep milk proteins under their native form.

The method of the invention takes advantage of membrane ultrafiltrationto effect fractionating of the components of the casein-based rawmaterials, but by combining said ultrafiltration step with an enzymatichydrolysis step.

A number of methods are known for the hydrolysis of proteins, e.g. milkproteins. In fact acid hydrolysis does allow obtention of freeamino-acids solutions, but will destroy some of the latter. Alcalinehydrolysis will preserve tryptophane, but cause an insolubilizationwhich substantially reduces the nutritive value of the initial proteinicconcentrates.

Enzymatic proteolysis has been known and used for quite a long while foranalytic or nutritional purposes, the main object being to solubilizethe proteins. Abundant reports will be found in literature on numerousalimentary uses of soybean protein hydrolyzates [see ARAI S., NOGUCHIM., KUROSAWA S., KATO H. and FUJIMAKI M. (1970) Applying proteolyticenzymes on soybean, 6-deodorization effect] of fish proteins: [see HEVIAP., WHITAKER J. R. and OLCOTT H. S. (1976)-Solubilization of a fishprotein concentrate with proteolytic enzymes. J. Agric. Food Chem. Vol.24 (2) 383-385] or of colza, by action of animal, microbial or vegetableproteases.

However, application of those techniques to milk proteins on acommercial scale is still quite limited.

Enzymatic proteolysis is free from the drawbacks of the chemicalprocesses. Conditions of hydrolysis are moderate and thus preserve thenutritional value of the products.

Generally, hydrolysis leads to peptides having a pronounced bittertaste. This feature acts to limit the use of such hydrolyzates for humanalimentation. The degree of bitterness of an hydrolyzate is mainlydependent on the nature of the proteic substrate and the enzymespecificity. To remove bitterness, it was suggested to use the action ofexopeptidases. See for example ARAI S., YAMASHITA M., KATO H., FUJIMAKIM. (1970) -Agric. Biol. Chem. 34, 729, as well as CLEGG K. M., SMITH G.and WALKER A. L. (1974) -Production of an enzymatic hydrolyzate ofcasein of a kilogram scale. J. Food Technol. 9, 425-431. Moreover, itwas proposed to modify the peptides by addition of glutamic acid beforethe plasteic reaction. It is also possible to proceed by removal of thehydrophobous amino-acids.

However, all these known techniques are unsatisfactory and unfit to meetthe requirements of the invention. In fact, an extensive solubilization,caused by the use of exopeptidase, will increase the amount of freeamino-acids and especially of arginine, lysine, tyrosine, valine,phenylalanine, methionine and leucine, and the net result thereof wouldbe to burden the systems for free amino-acids transport at theintestinal barrier, thus causing a reduction in the nutritionalefficiency of the hydrolyzates. On the other hand, the intrinsec qualityof the hydrolyzates will be modified since the amino-acid equilibriumitself is changed, this requiring additional provision of freeamino-acids.

From the technological standpoint, enzymatic hydrolysis is most usuallyeffected with a discontinuous reactor system. The enzyme is added to theproteinic solution to be treated. After a more or less prolongedresidence time, in conditions promoting enzymatic activity and substrateattack, the pH is modified and the enzyme is deactivated by a mildthermal treatment. Centrifugation may be effected to remove theundigested insoluble fraction. However, according to this technique ofdiscontinuous enzymatic hydrolysis reaction, it is difficult to use ahigh enzyme to substrate ratio. Now, it is known, see ROBINS R. C.(1978) - Effect of ratio of enzymes to substrate on amino-acid patternsreleased from proteins in vitro. Internat. J. Vit. Nutr. Res. 48, 44-52,that the enzyme/substrate ratio has a critical influence on the natureof the free amino-acids and peptides released during proteolysis. With adiscontinuous process, the enzymes must be destroyed at completion ofhydrolysis, when provided in excess as would be compulsory with theaforesaid high ratios.

It was also proposed to use reactors, with fixed enzymes. However, theseare attended by substantial drawbacks from the practical standpoint. Asa matter of fact, the optimum conditions for enzyme activity, especiallypH conditions, are shifting, so that the reactor operation is notsatisfactory at all times. Moreover, it occurs bacteriological problems,plugging of the fixation beds as well as protein adsorption onto thesubstrate. Moreover, the enzymatic reaction tends to get inhibited astime elapses, due to the formation of an enzyme-proteic fragmentcomplex. Inhibition may also be caused by the substrate nature. It ismoreover very difficult to use multi-enzyme systems because of phenomenaof enzyme competition with respect to the substrate and because enzymestability will vary with time.

The invention took advantage of means already known in some otherapplications and which consist in using enzymatic reactors provided withmembranes. Reference may be made for example, to the article by CHEFTELC. (1972) - Solubilisation enzymatique du concentre proteique depoisson. Essai de recyclage des enzymes. Ann. Technol. Agric. 21, (3)423-433 which describes a membrane-type reactor applied to proteolysisof fish proteic concentrates. The ultrafiltration membrane permits tokeep the enzyme in solution within the reactor, as well as the proteinicsubstrate. Only the hydrolysis products, i.e. the peptides, are removedas their formation proceeds. However, in practice, the use of such areactor is not easy, as pointed out by CHEFTEL. The substrate should becompletely solubilizable by the enzyme and the proteic solution has tobe of irreproachable bacteriological quality.

As documents showing the state of the art, the following references mayfurther be quoted:

French Patent 77,24 069 (publication No. 2,399,213) describes treatmentof an hydrolyzate by ultrafiltration, then electrodialysis. Thisdocument evidences the fact that it was known to ultrafiltrate a proteichydrolyzate. The method described in this patent makes it possible toproduce a pure solution of natural amino-acids;

French Patent 74,39 311 (publication No. 2,292,435) relates to theobtention of calcium phosphocaseinate from a milk ultrafiltrationretentate. The teaching of this patent has therefore for its objectproduction of calcium phosphocaseinate. It does not relate to thetreatment of monovalent cation phosphocaseinates or derivatives thereof;

The reference CHEMICAL ABSTRACTS, Vol. 87, No. 19, Nov. 7, 1977, page265, abstract 148 285 p COLUMBUS OHIO (U.S.) & J. Dairy Research, Vol.44, No. 2 (1977), pages 373-376, D. W. WEST "A simple method for theisolation of a phosphopeptide from bovine α s₁ -casein", describes theobtention of a phosphopeptide from caseinate. The method involvesenzymatic hydrolysis by trypsine, and fractionating steps by gelfiltration and chromatography, but it begins by a reaction with CNBrleading to quite specific products;

The reference CHEMICAL ABSTRACTS, Vol. 91, No. 21, Nov. 19, 1979, page523, abstract 173 597 g COLUMBUS OHIO (U.S.) & Enzyme Microb. Technol.,Vol 1, No. 2 (1979), pages 122-124, P. P. ROOZEN and al."Enzymaticprotein hydrolysis in a membrane reactor related to tasteproperties" describes hydrolysis in an enzymatic reactor, with a view toimprove the taste of proteic hydrolyzates. This document thus evidencesthe fact that the enzymatic reactor is a known apparatus.

According to the invention, the method of treatment is applied toproteic solutions free of bivalent ions, such as calcium and magnesium.As a matter of fact, there is essentially used a casein-based rawmaterial containing monovalent cation phosphocaseinates or derivativesthereof.

Broadly speaking, the method of the invention is characterized in thatthe above-defined raw material is subjected to enzymatic hydrolysis bymeans of at least one proteolytic enzyme able of reproducing the proteicdigestion occurring in vivo in the human body; the thus obtainedhydrolyzate is subjected to at least one ultrafiltration step onmembranes which allow all the peptides in the hydrolyzate to pass in thepermeate; the permeate is added with at least one bivalent cation saltcapable of forming aggregates with the phosphorylated fraction of saidpeptides, this leading to a solution which essentially containsaggregates of phosphopeptides and non phosphorylated peptides; andseparation is effected by at least one ultrafiltration step between thenon phosphorylated peptides and the phosphopeptides, the latter having alarger particle size, by bringing the solution into contact with atleast one membrane capable of retaining said phosphopeptides.

The casein-based raw materials liable to be treated by the method of theinvention contain monovalent cation phosphocaseinates, such as sodium,potassium or ammonium phosphocaseinates. The treatment may also beapplied to a raw material containing derivatives of saidphosphocaseinates, especially paracasein. All such compounds are knownto those skilled in the art and are obtainable by industrial means. Forexample, preparation of monovalent caseinates, such as sodium caseinate,first involves preparation of casein, starting e.g. from milk, byprecipitation at the isoelectric point. After washing said casein, thecasein precipitate is added with sodium hydroxide, potassium hydroxide,ammonium hydroxide or other basic compounds including monovalent ionsand adapted to resolubilize the casein. There is finally obtained aproteic solution containing said monovalent cation caseinates,preferably sodium and potassium caseinates. Such substances can bedirectly used as a raw material for the method of the invention.

In an alternate embodiment, use may be made of derivatives of saidcaseinates, in particular under the form of paracasein. For thispurpose, the solution of monovalent cation phosphocaseinates ispreviously treated by addition thereto of rennet, which causes anhydrolysis reaction. The hydrolysis product will contain paracaseinates,and the caseinomacropeptides (CMP). The paracasein is then subjected toprecipitation by any known means, preferably by acidification to pH 4.6with any organic or inorganic alimentary or medical acid, e.g.hydrochloric acid, phosphoric acid, sulphuric acid, acetic acid, lacticacid or other similar acids. In practice, hydrochloric acid ispreferred. Then, the supernatant solution, containing thecaseinomacropeptides, is separated from the precipitated paracasein. Thelatter is used in turn as a raw material for the method of theinvention. In this modified embodiment, the solution obtained containscaseinomacropeptide which may constitute a valuable product. To purifyand separate said product, said solution may be neutralized with a basiccompound, such as sodium hydroxide. The CMP can be prepared under aconcentrated form by ultrafiltrating the solution, after additionthereto of calcium chloride.

In a preferred form of the above described alternate embodiment, thesodium caseinate in water solution (3%) was hydrolyzed by rennet (20ml/100 l). The paracasein was then precipitated by acidification (to pH4.6) with HCl. The supernatant solution containing thecaseinomacropeptide was thenafter neutralized (to pH 7.0) with sodiumhydroxide, and concentrated by ultrafiltration after being added with0.5 g of CaCl₂. Upwards of 1000 liters of 3% caseinate solution, thisalternate embodiment allows obtention of about 30-40 liters of 3%caseinomacropeptide solution.

Irrespective of the raw material used in the method of the invention,the initial step consists in an enzymatic hydrolysis with at least oneproteolytic enzyme able of reproducing the proteic digestion occurringin vivo in the human body. As previously mentioned, such an hydrolysisis effected to advantage in a device which combines an ultrafiltrationequipment with an enzymatic reactor, this permitting continuousoperation.

In such an embodiment, the enzymatic hydrolysis step is effectedcontinuously by feeding the casein-based raw material to a reaction zoneto bring it into intimate contact with the enzyme, the reaction productis withdrawn continuously and transferred from the reaction zone to anultrafiltration zone, wherefrom there is withdrawn, also continuous by apermeate which forms the peptidic hydrolyzate.

During the enzymatic hydrolysis step, the pH should be adjusted in therange of 7 to 9. For this purpose, there is fed in continuous ordiscontinuous manner, into the reaction zone, a basic compound which maybe sodium hydroxide or carbonate, potassium hydroxide or carbonate,ammonium hydroxide or a mixture thereof. The selection of a particularbasic compound will depend on the intended purpose for the finalproduct.

As an enzyme, use is made preferably of at least one proteolytic enzymecapable of reproducing the proteinic digestion which occurs in vivo inthe human body. Therefore, use may be made to advantage of pancreatin,which is a complex mixture containing trypsine, chymotrypsine and othersecondary proteolytic enzymes. In practice, it may be resorted to anatural pancreatic extract commercially available and readilyobtainable. However, if so required, use may also be made of enzymesformed by a synthetic mixture, e.g. of alpha-chymotrypsine and trypsine.Preferably, the synthetic mixture used has a composition approximatingthat of pancreatin, and therefore including the secondary enzymescontained in natural pancreatic extract. It was found according to theinvention that at a pH ranging from 7 to 9, and preferably from 7 to8.5, e.g. of 8, pancreatin and other similar enzymes meeting therequirements of the invention have maximum stability.

It is further advisable to comply with rather strict temperatureconditions in the enzymatic hydrolysis zone. In fact, it was found thatenzyme activity was more strongly influenced by the temperature than bythe pH. In particular, tests have shown, according to the invention,that with trypsine, the maximum temperature during enzymatic hydrolysisshould not be higher than 54° C., and that with chymotrypsine, saidtemperature should not be higher than 45° C. In practice, when use ismade of pancreatin, a compromise will be made taking into account boththe optimum conditions for intestinal proteolysis in vivo (temperatureof the order of 37° C.) and the fact that higher temperatures are lessfavorable to germ growth and allow for higher ultrafiltration outputs.In general, the selected temperatures are of the order of 37° to 40° C.,and most preferably still close to 37° C.

Obviously, the reaction parameters, viz. the pH and the enzymatichydrolysis temperature, are interrelated. Thus, it will fall to thoseskilled in the art to select the most favourable conditions in eachparticular case.

To effect optimum enzymatic hydrolysis, it is also advisable to selectcarefully the ultrafiltration membrane to be used in conjunction withthe enzymatic reactor. The membranes used may be of any organic orinorganic type. A membrane structure which afforded good results is thatof modules with hollow fibers. As a guidance, use may be made of themembranes of Societe AMICON available under the trade name H10P5(cut-off threshold 5000) and H10P10 (cut-off threshold 10,000) as wellas membranes of Societe ROMICON available under the trade name PM 2(cut-off threshold 2000) or PM50 (cut off threshold 50,000). The onlyrequirement to be met is that, in operation, the membrane should retainefficiently the enzyme, while having satisfactory performances,especially as regards its life time.

The method of the invention may be carried out in two separate stages: afirst stage consisting in the enzymatic hydrolysis step, and a secondstage consisting in the ultrafiltration step associated with saidhydrolysis step. The equipments for carrying out each of these steps canbe separate or integral. However, as an alternate embodiment, the methodmay also be performed continuously, both aforesaid stages being effectedin a single apparatus. During the initial operating period, e.g. forabout one hour, the permeate (liquid flowing through the membrane) isrecycled to the hydrolysis zone for the obtention of the desired degreeof hydrolysis of the casein-based material. After hydrolysis, thereactor is fed with the casein-based raw material to be treated at aflow-rate identical with that of the permeate.

Thus, a preferred embodiment of the invention consists in combining andcarrying out continuously the enzymatic hydrolysis step and the membraneultrafiltration step, whereby all the peptides in the hydrolyzate can berecovered in the hydrolyzate. The ultrafiltration membrane used inconjunction with the enzymatic hydrolysis step should have suchcharacteristics as to allow free passage of all the peptides in thehydrolyzate. Membranes having a cut-off of 50,000 or more proved to besuitable.

According to an essential feature of the invention, the permeate is thenadded with at least one bivalent cation salt able to form aggregateswith the phosphorylated fraction of the peptides. Indeed, it was foundthat by complexing the bivalent cations, inter-aggregation of thephosphopeptides is facilitated, whereby the latter can be separated fromthe non phosphorylated peptides. Separation between the phosphopeptidesand the non phosphorylated peptides obtained upon enzymatic hydrolysisis based on the ability of phosphoserines to complex alkaline-earthions, particularly calcium and magnesium ions. When, as is the case forthe method of the invention, hydrolysis is effected on proteic solutionsfree of calcium and/or magnesium, it is important to add to the peptidicsolutions obtained upon hydrolysis the involved bivalent cations, whichexert the complexing function.

In practice, it is preferred to use, as complexing bivalent cations,calcium cations, for example brought in by calcium chloride. The amountof agent for complexing the bivalent cation-based peptides which,according to the invention, should be added to the peptidic solution isnot critical. Practically, calcium chloride amounts of the order of 0.5%by weight, as related to the peptide solution, were deemed suitable.Obviously, it rests with those skilled in the art to select the bivalentcompounds and amount thereof to be used, also taking into account thefeatures of the subsequent stage of separation between thephosphopeptide aggregates and non phosphorylated peptides, saidseparation being effected, according to the invention, by anultrafiltration step. In other words, due attention should also be paidespecially to the cut off threshold of the ultrafiltration membrane, soas to avoid passage of the phosphopeptidic aggregate through thismembrane.

According to an embodiment which afforded good results, there is used inassociation with the complexing agent a mineral phosphate, such assodium acid phosphate PO₄ HNa₂. The presence of such a phosphatecompound may enhance the complexing action and formation of largephosphopeptidic aggregates. However, in some cases and especially whenit is desired to obtain, at the end of the treatment, phosphopeptidesfractions without exaggerated mineral phosphate enrichment, then theamount of phosphate added may be reduced, or even completely suppressed,provided that there is used at the final stage of the process a membranecapable of retaining the phosphopeptides and with a cut-off thresholdranging preferably between 2000 and 50,000, and more preferably between2000 and 10,000.

As previously mentioned, each of the above ultrafiltration steps may befollowed by a diafiltration step during which there is added,continuously or discontinuously, a liquid such as water or aqueoussalt-containing solution, with a view to further purify theultrafiltration products. In the method of the invention, water provedto be suitable for diafiltration.

As a result of the ultrafiltration and diafiltration steps which followthe enzymatic hydrolysis step, there is obtained, on the one hand, apeptidic solution which is subjected to further treatment by the methodof the invention, and on the other hand a fraction (retentate) whichconsists of proteic residue and residual enzymes. As a result of theultrafiltration and diafiltration steps effected at the final stage ofthe method of the invention, there is obtained on the one hand, as apermeate, non phosphorylated peptides, and on the other hand; as aretentate, phosphopeptides.

In a preferred embodiment, preparation of the peptidic fractions waseffected starting from a 6% sodium caseinate solution. Hydrolysis in theenzymatic reactor was done with pancreatin in an amount of 4 g/l at pH 8and at 37° C. The reactor content was then diafiltrated with water. Thepeptide solution was thereafter acidified to pH 6.2, aggregation beingcaused by addition of CaCl₂ (0.5%) and PO₄ HNa₂ (0.2%). The peptidesolution then undergone ultrafiltration and diafiltration. Starting from1000 liters of 6% sodium caseinate, there may be obtained 900 liters ofnon phosphorylated peptidic solution (45 g/liter) and 100-120 liters ofphosphopeptide solution (80 g/liter).

The phosphopeptides thus obtained at the final stage of the method ofthe invention constitute the most interesting valuable product. Indeed,the latter has a high phosphoserine content and contains low amounts ofaromatic amino-acids (phenylalanine, tyrosine, tryptophane).

The thus obtained fraction, rich in phosphopeptides, may therefore becharacterized both by its particular composition in amino-acids and by ahigh content of mineral matter (ashes) with respect to the totalnitrogen, as the phosphopeptidic fraction acted to complexe the addedsalts.

Table I hereunder shows the main characteristics of the products of theinvention, those of the monovalent caseinate being set forth in thefirst column, as a reference.

                  TABLE I                                                         ______________________________________                                                          Non                                                                   K or Na phosphorylated                                                                             Phospho-                                                 caseinate                                                                             peptides     peptides                                       ______________________________________                                        Total amount of                                                                            12%      >12%         <4%                                        aromatic amino-                                                               acids (Tyr, Phe,                                                              Trp)                                                                          Serines     4.9%       <4%         <20%                                                                          >8%                                         ##STR1##   <0.01     <0.02        >0.2                                       Free amino-acids                                                                          --        <10%         <3%                                        ______________________________________                                         (1) N.sub.T = total nitrogen × 6.38                                

The phosphopeptides obtained by the method of the invention are suitablefor numerous applications in the alimentation field.

The products of the invention are useful for alimentation, in particularhuman alimentation, and for therapeutic nutrition. It is known indeedthat, in human milk, the so-called organic phosphorus, i.e. bound to theproteins and bound to the lipids, is comparatively more abundant than inother milks, especially cow's milk. Thus the ratio ##EQU1## is of about0.83 in human milk as against 0.34 in cow's milk.

More precisely, the ratio of organic phosphorus bound to the nitrogenversus inorganic phosphorus is about 0.70 in human milk against 0.36 incow's milk.

Therefore, the products of the invention will found applications in thefield of so-called milk maternisation.

But, in general, it is admitted that the main good property of woman'smilk proteins is to ensure a remarkable nitrogen anabolism, togetherwith renal osmotic load and a H⁺ ion load of particularly low values.

Now, this conjunction of a very high nitrogen anabolism with low renalosmotic load and H⁺ ion-load is particularly sought in the fields ofreanimation and therapeutic nutrition, where high anabolism requirementsand functional renal deficiency are quite often coexistent.

The products of the invention are suitable to meet those requirements.For certain applications, the products of the invention contain aninsufficient amount of some essential amino-acids (phenylalanine,tyrosine, tryptophane, cystine). They may then be associated toadvantage with other proteins or peptides or either alpha-keto-acid oralpha (OH) acid homologues of essential amino-acids, for restoring of agood amino-acid equilibrium leading to optimal biological value.

It will also be noted that such products (phosphopeptides) have a highaffinity for macroelements (calcium, magnesium) and for oligoelementsas, particularly, iron, zinc, copper, chrome, nickel, cobalt, manganeseand selenium.

The phosphopeptides according to the invention can advantageously beconverted into salts of said elements by usual means. Thus, to obtainsuch an organophosphorated salt, it may be used, as a diafiltrationsolution for purifying the phosphopeptides, a solution of a saltcontaining the element to be introduced, e.g. a solution of ironchloride in the case of iron. These organophosphorated salts are highlysoluble and they may advantageously be used as carriers for theparticular elements.

The products of the invention meet the nutritional requirements ofpatients suffering from pancreatic deficiency, metabolic diseases,nutritional deficiency or distress, which may be or not associated witha functional or organic renal deficiency, in particular when they areassociated with peptides, essential amino-acids or essential amino-acidhomologues.

The invention therefore finds a direct application in dietetic alimentsor therapeutic nutriments which are perfectly assimilable by the humanbody.

Irrespective of the source of proteins, peptides or amino-acids used,these phosphopeptides permit to regulate, in the most desirable manner,the amount of organic phosphorus bound to nitrogen in the formulation tobe created.

As previously mentioned, the phosphopeptides according to the inventionand their derivatives, notably the organophosphorated salts which theyform with the mineralmacroelements, such as calcium and/or magnesium,and/or with oligoelements (Fe, Zn, Cu, Cr, Ni, Co, Mn, Se for instance)find an interesting application in the dietetic.

The invention concerns therefore dietetic compositions containing anefficient quantity of at least one such phosphopeptide or derivative ofphosphopeptide in association with a carrier acceptable from thenutritional point of view. Such an efficient quantity may vary in widelimits according to the effect seeked. For information purpose, aquantity in weight of 10% with respect to the total of the compositionis suitable in the usual cases.

The products of the invention can also be applied as such, asmedicaments for the man and the animal.

The medicaments concerned are appropriated to alleviate all diseasesinvolving a lack of organic phosphorus and of certain mineral elements.For illustration purpose and not at all limitative, some specificexamples of such application are hereafter given.

The mineral derivatives of phosphopeptides according to the invention,consisting of their calcium salts, constitute a protidic mineralsupplement rich in organic phosphorus and in calcium. They find anapplication as medicaments, for instance in the following cases:

recalcification of the bones after fracture,

osteoporosis treatment

calcic addition during treatment of rickets.

The derivatives of phosphopeptides according to the invention,consisting in their manesium salts, constitute a mineralo-protidicsupplement rich in organic phosphorus and in magnesium. They find anapplication as medicaments to remedy to all the forms of magnesicdeficit, more particularly to the adult, for instance in the followingcases:

needs in Mg greatly increased by the stress

bad use of Mg food by the old people

increase of Mg-needs to the pregnant woman.

Medicaments containing derivatives of phosphopeptides consistigng inmixt calcium and magnesium salts, are used in the same way asmineralo-protidic supplement. It goes without saying that similarapplications can be foreseen of varied phosphopeptides salts accordingto the invention, although, in practice, calcium and/or magnesium saltsare preferred.

It is to be noted that the phosphopeptides, such as they are obtainedaccording to the process of the invention are under the form of theirsalts of bivalent cations, particularly salts of calcium and/ormagnesium. If necessary, such salts can be converted into neutralphosphopeptides by lowering the pH of the medium for example to 4.6about, but in practice, this procedure is not compulsory, because thephosphopeptide salts are perfectly suitable to be used as such. Thus inthe conditions of use, and more particularly when the phosphopeptidesare included in dietetic or pharmaceutical compositions, they are foundunder the form of salts, for instance of calcium and/or magnesium.

The macroelements (preferably calcium and/or magnesium) can be replaced,at least partially, by oligoelements.

The derivatives of phosphopeptides according to the invention whichcontain oligoelements find an application corresponding to that ofparticular oligoelements.

The general indications of medicaments containing phosphopeptides andoligoelements derivatives are among others the digestive bad absorptionsinducing lack of oligoelements (Fe, Zn, Cu, Cr, Ni, Mn, Se). Saiddigestive bad absorptions appear more particularly during inflamatoryileitis, in the case of resect bowels, celiac diseases and radic bowels.As examples, the lack of zinc can cause acrodermatitis enteropathica,diarrhoeas, an increased sensitiveness to infections, hypogonadism.Lacks of iron can entail sideropenic anemia.

The medicaments according to the invention are preferred for thetreatment of lack of zinc, copper, chromium and iron.

The invention also concerns pharmaceutical compositions containing aproduct of the invention in admixture with the usual excipients. Takinginto account the physical form of the new product (soluble powder inaqueous medium), the form of presentation does not raise any difficulty.The new products can be ingested or given as such, especially by enteraltract, for instance mixed with usual food. They can also be presentedunder the form of compositions with usual excipients for instancesuitable by oral administration. Appropriated compositions according tothe invention can thus be presented under the form of tablets orcapsules, with known excipients such as talcum, magnesium stearate,finely divided silica, and any other similar carrier known by the manskilled in the art.

As example, it has been indicated hereunder a particular caseillustrating the preparation of pharmaceutical compositions foradministration by oral route. Tablets have been prepared in the usualmanner starting with the following formulation:

phosphopeptide (or phosphopeptide salts) according to the invention: 200mg

excipient QS for a tablet terminated at: 300 mg

The excipient used can be talcum, magnesium stearate or the silicaavailable on the market under the denomination "aerosil".

Capsules dosed at 100 mg of phosphopeptide (or salt) according to theinvention and containing an usual excipient QS for a capsule terminatedat 200 mg have been prepared in the same manner.

The invention will now be illustrated by no way of limitation by thefollowing description and examples given hereunder.

The description will be made with reference to the appended drawings,wherein:

FIG. 1 is a flow-sheet illustrating the method of the invention.

FIG. 2 is a diagram of an enzymatic reactor suitable for use in themethod of the invention.

As shown in FIG. 1, the monovalent (sodium or potassium) caseinate-basedraw material A can be subjected either directly to the process(reference 1) or to a previous treatment through the alternative path 2.According to this alternative embodiment, the raw material is hydrolyzedwith rennet to form a solution containing the corresponding (sodium-orpotassium-)paracaseinate and the caseinomacropeptide. The paracasein isprecipitated by acidification (HCl). Then, separation is effectedbetween the precipitated paracasein and the caseinomacropeptide. Thelatter (CMP) constitutes a by-product G of the method which, while thosesteps are not shown, may be neutralized, e.g. by sodium hydroxide, andthereafter concentrated by ultrafiltration after beind added withcalcium chloride.

Therefore, the raw material for the method is either caseinate A or itsderivative, viz. paracasein.

The initial stage of the method is an hydrolysis (reference 3) in anenzymatic reactor. In the drawing, there is shown addition of a baseadapted to raise the pH to 8, so that the hydrolysis will proceedsatisfactorily with a proteolytic enzyme such as pancreatin. Thehydrolysis product is thereafter subjected to an ultrafiltration (4),then to a diafiltration (5) with water. There is obtained both apeptidic fraction E, and a residual fraction F containing the residualproteins and enzyme. It is the peptidic fraction E which undergoesfurther processing in the method. The subsequent stage is a complexingstep (reference 6) during which fraction E is added with calciumchloride and possibly with a phosphate (PO₄ HNa₂). Upon complexing oraggregation of the peptides, the product is subjected to anultrafiltration 7, then a diafiltration 8 with water. There is thusfinally obtained a fraction B (permeate) which is enriched in nonphosphorylated peptides, and a fraction C which is enriched inphosphopeptides.

FIG. 2 shows a membrane-type enzymatic reactor which may used in themethod of the invention.

Said reactor includes first a reaction tank generally designated byreference 15. Continuous feeding of phosphocaseinates occurs throughduct 16. A device 17 serves both to measure the pH and to keep itconstant in the reaction tank by neutralizing the H⁺ ions released whenthe peptidic bonds are broken. Said device was a Mettler pH-statcomprising a potential amplifier, an equivalence point presetting switchand an automatic burette for feeding the reactive, the latter being abasic compound such as mentioned above. No excessive electrode foulingwas noted. The hydrolysis product withdrawn from the reaction tankthrough duct 18 is conveyed by an automatic membrane-type pump 19. Apractical example is the pump of AMICON LP 20 A model, with an output of720 l/h at about 25 psi. At the outlet of the pump, the product flowsthrough a duct 20 and is fed onto a pre-filter 21 having a pore size of150 microns. Reference 22 designates the ultrafiltration module. In aspecific example, the system used was AMICON LP 10 S model havingultrafiltration cartridges of the hollow fiber type. The permeate wasrecovered through a duct 23 and formed the desired peptidic hydrolyzate.The retentate was withdrawn from module 22 through duct 24, then fed toan exchanger 25 and conveyed through duct 26 to be recycled intoreaction tank 15.

The membranes used were of the hollow fiber type having the followingcharacteristics:

    ______________________________________                                                            surface                                                   Type        cut-off area        Manufacturer                                  ______________________________________                                        H 10 P 5    5000    0,9 m.sup.2 AMICON                                        H 10 P 10   10,000                                                            PM 2        2000    1,4 m.sup.2 ROMICON                                       PM 50       50,000                                                            ______________________________________                                    

EXAMPLE 1

In this example, sodium caseinate is used as a raw material.

The caseinomacropeptide and peptidic fractions according to the processshown in FIG. 1 were prepared in two stages:

(1) Sodium caseinate in solution in water (3%) was hydrolyzed in a tankwith rennet (20 ml/100 liters-BOLL rennet, 1/10,000) at 37° C. at pH 6.8during 50 minutes. The paracasein was then precipitated by acidificationto pH 4.6 with hydrochloric acid 4N; 440 ml of acid were necessary for100 l serum.

After settling, the supernatant containing the CMP was filtrated on agauze, then centrifuged (1000 g during 8 min.) after pH re-adjustment to6.6 with potassium hydroxide 2N. This solution was thereafterconcentrated by ultrafiltration on a membrane, after being added withCaCl₂ (0.5 g/l). The apparatus used was an Amicon module of type DC 10equipped with Romicon membranes of Hollow Fiber type XM 50 having asurface area of 1.4 m².

The chemical analysis of the various products obtained in thisproduction run are set forth in tables II and III.

                  TABLE III                                                       ______________________________________                                        Composition in amino-acids (g of amino acid for                               100 g)                                                                        ______________________________________                                        Asp      6.9           Gly  1.4      Ile  10.3                                Thr      9.6           Ala  6.1      Leu  3.5                                 Ser      3.1           Cyst ε                                                                              Tyr  ε                           Glu      23.2          Val  8.7      Phe  1.7                                 Pro      12.7          Met  1.5      Lys  6.6                                                                      His  0.6                                                                      Arg  0.9                                 ______________________________________                                    

(2) The sodium caseinate in solution in water (6.2%) was hydrolyzed in amembrane enzymatic reactor identical with that shown in FIG. 2. Themembranes used were of the Hollow Fiber type XM 50, having a surfacearea of 4.9 m². The enzyme (Sigma pancreatin of bovin origin having anactivity 4 NF) was added at a concentration of 4 g/liter. The reactor pHwas kept at 8 by addition of potassium hydroxide 2N. Hydrolysisproceeded at 37°-40° C. Before being collected, the permeate wasrecycled in the tank during one hour. The thus obtained permeatecontained the phosphopeptides and non phosphorylated peptides. Thepermeate was acidified to pH 6.4, then phosphopeptide aggregation wascaused by addition of CaCl₂ (0.6% and of PO₄ HNa₂ (0.1%). Thereafter,fractionating of the two groups of peptides was effected byultrafiltration and diafiltration with water so as to remove the wholenon phosphorylated peptide fraction. The concentration and diafiltrationsteps were carried out on membranes of XM 50 type at pH 6.5 and at atemperature of 8° C. The diafiltrated concentrate obtained correspondedto the phosphopeptidic fraction.

                                      TABLE II                                    __________________________________________________________________________                                NPN*                                                              Dry matter                                                                          N.sub.T × 6.38                                                                TCA 6%                                                                             TCA 12%                                      __________________________________________________________________________    Starting caseinate (A)                                                                        31.2  27.1  --   --                                           Caseinate after rennet action                                                                 --    --    182.5                                                                              95.4                                         Renneted lactoserum                                                                           3.45  1.88  166.8                                                                              105.8                                        after centrigufating, pH re-                                                  adjustment and CaCl.sub.2 addition                                            Lactoserum concentrated by                                                                    31.3  30.1  2649 1191                                         ultrafiltration or CMP(G)                                                     Ultrafiltration permeate                                                                      2.07  0.3%  --   --                                           __________________________________________________________________________     Chemical composition of the products as expressed in g/kg, except for non     proteic nitrogen (NPN) expressed in ppm of nitrogen                      

The analysis of the various products are shown in tables IV and Vhereunder:

                  TABLE IV                                                        ______________________________________                                        Chemical composition of the products obtained                                            Dry matter                                                                            N.sub.T × 6.38                                                                     Ca     Ashes                                               g/kg    g/kg       g/kg   g/kg                                     ______________________________________                                        (A) Starting 62.3      54.2       --   --                                     caseinate                                                                     (E) Total    62.3      45.9       --   --                                     peptides                                                                      (B) Non phospho-                                                                           53.7      47.3       --   --                                     rylated peptides                                                              (C) Phosphopeptides                                                                        15.0      7.90       2.35 6.3                                    ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        Compositions of the amino-acids in the phosphopeptides                        (g of amino-acid per 100 g)                                                   ______________________________________                                        Asp        5.6               Cys  ε                                   Thr        3.3               Met  0.45                                        Ser        18.5              Ile  9.5                                         Pro        4.7               Leu  2.9                                         Glu        36.8              Tyr  ε                                   Gly        1.7               Phe  ε                                   Ala        2.8               Lys  0.9                                         Val        8.7               His  0.5                                                                      Arg  0.3                                         ______________________________________                                    

Illustrative examples relating to the applications of the products ofthe invention will be given hereunder, while implying no limitation.

EXAMPLE 2

This example relates to a reanimation product intended for:

enteral administration to patients requiring a proteic input of about 7to 15% of the total calorific input (TCI), for instance in the followingailments mucoviscidose or cystic pancreas fibrosis, renal deficiency,patients having an infectious or inflammatory disease of the intestinalmembrane nutrition distress requiring intense anabolism with reducedrenal osmotic load and H⁺ ion load. These proteins are preferablybrought into a pre-digested state.

EXAMPLE OF CENTESIMAL FORMULA ACCORDING TO THE INVENTION

    ______________________________________                                        Phosphopeptides from                                                                         4 to 8%                                                        Lactoserum peptides                                                                          60%                                                            Casein peptides from                                                                         32 to 36%       2.50 g                                         CMP from       0 to 4%                                                        Lipids:                                                                       mixture in equal parts of:                                                    butter oil     0.5    g                                                       mean chain triglyceride                                                                      0.5    g                                                       (MCT)                                                                         maize oil      0.5    g          4.10 g                                       sunflower oil  0.5    g                                                       glycerol monostearate                                                                        2.1    g                                                       glucids:                                                                      glucose polymers                                                                             10     g                                                       glucose        1.5    g          13.00 g                                      galactose      1.5    g                                                       vitamins:                                                                     A, D, E, B.sub.1, B.sub.2, PP, B.sub.5, B.sub.6                                                          according to                                       B.sub.12, folic acid, Biotine,                                                                           FAO/MWO                                            C vit.                     recommendations                                    mineral elements                                                              (calcium, sodium, potassium,                                                  magnesium, phosphorus, zinc,                                                  iron, copper, manganese,   0.455 g                                            chlorine, iodine)                                                             distilled water                                                                              as required for 100 g                                          ______________________________________                                    

EXAMPLE 3

This example relates to a reanimation product for enteral administrationto patients requiring a proteic input of about 12-25% of the TotalCalorific Input, under the form of proteins. Such alimentation issuitable for any situation necessitating substantial nitrogen anabolismand an oversupply of organic P⁻.

EXAMPLE OF CENTESIMAL FORMULA

    ______________________________________                                        mixture of small peptides according                                           to the invention.                                                             phosphopeptides from                                                                          8 to 28%                                                      lactoserum peptides from                                                                     22 to 60%         3 to 6.25 g                                  casein peptides from                                                                         12 to 70%                                                      lipids:                                                                       T.C.M.         2.30      g                                                    Oil very rich in                                                                             0,50      g                                                    essential fatty acids              2.90 g                                     Emulsifier     0.10      g                                                    glucids:                           12.25 to 15.5 g                            Small glucose                                                                 polymers from  8.25 to 10.5                                                                            g                                                    Glucose from   2 to 2.5  g                                                    Galactose from 2 to 2.5  g                                                    vitamins:                                                                     A, D, E, B.sub.1, B.sub.2, PP, B.sub.5,                                                                          according to                               B.sub.6, B.sub.12, folic acid,     FAO/MWO re-                                Biotine, Vit. C                    commendations                              mineral elements:                                                             (calcium, sodium, potassium                                                   magnesium, phosphorus, zinc                                                   iron, copper, manganese,     0.455 g                                          chlorine, iodine)                                                             distilled water                                                                              as required for 100 g                                          ______________________________________                                    

EXAMPLE 4

This example relates to a reanimation product for enteral administrationto patients requiring a proteic input of the order of 7-12% of theT.C.I., in particular in the following ailments:mucoviscidose or cysticpancreas fibrosis, renal deficiency, patients having an infectious orinflammatory disease of the intestinal membrane, nutrition distressrequiring intense anabolism with a reduced renal osmotic overload and areduced H⁺ ion load. These proteins are preferably brought into apre-digerted formula.

EXAMPLE OF CENTISIMAL FORMULA

    ______________________________________                                        mixture of small peptides according to the invention:                         phosphopeptides    4%                                                         casein peptides   36%                                                         lactoserum peptides                                                                             40%           2.50 g                                        CMP               20%                                                         lipids:                                                                       mixture in equal parts of:                                                    butter oil        0.5   g                                                     MCT               0.5   g                                                     maize oil         0.5   g         4.10 g                                      sunflower oil     0.5   g                                                     glycerol monostearate                                                                           2.1   g                                                     glucids:                                                                      glucose polymers  10    g                                                     glucose           1.5   g         13.0 g                                      galactose         1.5   g                                                     vitamins:                                                                      A, D, E, B.sub.1, B.sub.2, PP, B.sub.5, B.sub.6, B.sub.12                                                      according to                                                                  FAO/MWO                                     folic acid, Biotine, C vit.       recommendations                             mineral elements:                                                             (calcium, sodium, potassium,                                                  magnesium, phosphorus, zinc,                                                  iron, copper, manganese,          0.455 g                                     chlorine, iodine)                                                             distilled water   as required for 100 g                                       ______________________________________                                    

The peptide mixture containing the phosphopeptides according to theinvention may include to advantage the peptides obtained according tothe method described in French Patent Application filed on June 26, 1979under No. 79 16 483 in the name of INSTITUT NATIONAL DE LA RECHERCHEAGRONOMIQUE for "Hydrolysat enzymatique total de proteines delactoserum, obtention et applications". It will be recalled that such apeptidic hydrolyzate contains substantially no residual proteins andthat 50% of the peptide contain 2 to 5 aminoacids. More particularly,the hydrolyzate contains 70 to 90% of the nitrogen present under theform of peptides having a number of amino-acids lower than 10. Under aspecific form, the hydrolyzate corresponds to the following aminogram.

    ______________________________________                                        Aminogram:                                                                    ______________________________________                                               Ile  6.0              Arg  2.7                                                Leu  9.9              His  1.7                                                Lys  9.2              Ala  4.9                                                Cys  1.8              Asp  9.5                                                Phe  3.2              Glu  7.6                                                Thr  6.7              Gly  1.7                                                Tyr  3.6              Pro  6.2                                                Trp  2.0              Ser  5.5                                                Val  5.5              Met  2.0                                         ______________________________________                                    

The process for obtaining such an hydrolyzate consists in effectingfirst ultrafiltration of the lactoserum, then enzymatic hydrolysisthereof, and is more particularly characterized in that theultrafiltration retentate is contacted with a proteolytic enzyme able toreproduce the proteic digestion occurring in vivo in the human body,said enzyme being preferably pancreatin, the hydrolysis step beingcarried on until the product contains no more residual proteins, i.e. isfree of any nitrogen which can be precipitated by trichloroacetic acid,12%, then recovering the thus obtained hydrolyzate, which constitutesthe total enzymatic hydrolyzate desired.

What is claimed is:
 1. Essentially pure casein phosphopeptide salts ofcalcium, magnesium or both, comprising:(a) less than 4% by weight of atotal amount of aromatic amino acids, (b) a serine amount which isgreater than 8% by weight and less than 20% by weight, (c) a free aminoacid content of less than 3%, and (d) a ratio of: Ca+Mg+P/N_(T) which isgreater than 0.2, wherein N_(T) is the total amount of nitrogen×6.38. 2.The casein phosphopeptide salts of claim 1, wherein said aromatic aminoacids are phenylalanine, tyrosine and tryptophan.
 3. The caseinphosphopeptide salts of claim 1, wherein said salt is the calcium salt.4. The casein phosphopeptide salts of claim 1, wherein said salt is themagnesium salt.
 5. The casein phosphopeptide salts of claim 1, whereinsaid salt is the calcium and magnesium salt.
 6. The caseinphosphopeptide salts of claim 1, in combination with a pharmaceuticallyacceptable excipient.